The alpha subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the sigma subunit (the -10 and -35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, lambda pR, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to the lac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to approximately 90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP alpha subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (-10, -35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.